The role of protein kinases in regulating metabolism in lens is being addressed by studying: 1) the protein kinases, and 2) the endogenous proteins that serve as substrates for the lens protein kinases. The focus of this study is on the purification of the protein kinases and the characterization of the phosphorylation of four endogenous substrates by cAMP-dependent protein kinases. The phorphorylated proteins are Alpha-crystallin and 26K and 19K intrinsic membrane proteins. Comparison of the amino acid compositions of the 26K and 19K proteins suggest they are closely related. Detailed structural studies are in progress to determine how similar they are. Compounds that are thought to regulate the function of the 26K protein in vivo modulate the phosphorylation of the protein in vitro. Oxidative changes of lens proteins are thought to occur with aging and to contribute to the development of catarats. The goals of this project are to determine: 1) the extent of oxidative modification of crystallins and metabolic enzymes in both normal and cataractous lenses; 2) the nature of the modifications and mechanisms leading to the changes; 3) the effect of the modifications on structure and function of lens protein. Bovine and human lenses were used. Incubation of crystallins with ascorbate - FeC1-3-02 caused nondisulfide crosslinking of alpha and beta and the partial degradation of all 3 crystallin fractions. Conversion to more acidic species occurred with all 3 fractions, and nontryptophan fluorescence was produced in BetaH fraction. Longer incubations of homogenate with ascorbate - Fe-O2 mimicked changes similar to those in brunescent lenses. Proteins became brownish, insoluble and there was an increased carbonyl content.